Overview of Virus Life Cycle

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Overview of virus life cycle

cell recognition release from and internalization cells

progeny virus assembly

membrane breaching capsid disassembly replication and and genome release translation nucleus

Credit: Internet resources Replication scheme

Class IIa Class IIb +DNA ±DNA -DNA Class I

Class VI +RNA

Class IV mRNA -RNA Class V

±RNA

Baltimore classiﬁcation Class III Replication overview

Similar to host DNA – leading and lagging strand synthesis, primer required (RNA), DNA polymerase and other host factors required

lnicole3.blogspot.com Replication overview

Involvement of several enzymes 1) DNA unwinding protein

2) RNA polymerase

3) DNA polymerase III 4) DNA polymerase I (+ exonuclease activity)

5) DNA ligase 1 mistake/109 - 1010 bp replications Replication of animal DNA viruses

Enzymes mostly of host origin, some viruses carry their own enzymes Host machinery (enzymes etc) for generating DNA are in the nucleus (and mitochondria). Viruses utilizing host enzymes need to enter the nucleus. Viruses with own enzymes can replicate in the cytosol. Nuclear DNA viruses Cytoplasmic DNA viruses

I. Parvoviruses I. Poxvirus

II. Polyomaviruses

III. Adenoviruses IV. Herpesviruses V. Hepnadviruses

May carry some enzymes Example of class I: Polyomaviruses Example of class I: Polyomaviruses

Small, icosahedral, ~ 40 nm in diameter Contains circular duplex DNA Replicates in the nucleus DNA associates with host cell histones Utilizes host cell machinery mostly, except some viral proteins Viral proteins required for replication (enzymes, regulatory proteins etc) are expressed ﬁrst (early genes) Host RNA polymerase II recognizes promoter, mRNA produced Post-transcriptional RNA processing carried out by host enzymes Alternative processing of primary transcript produces small and large T antigens (Tag) Example of class I: Polyomaviruses Example of class I: Polyomaviruses

Replication starts in the late phase of infection Similar to host DNA replication process Host enzymes are used Host DNA polymerase recognizes SV40 origin of replication in the presence of large T antigen Host histones bind to newly synthesized viral DNA Example of class I: Adenovirus

Large non-enveloped icosahedral virus (~ 70 nm diameter) Linear, ds DNA, associated with virally coded basic proteins (not host cell histones)

Example of class I: Adenovirus

Early gene expression (proteins required for replication) carried out with host RNA pol and RNA modiﬁcation enzymes Encodes its own DNA polymerase for replication Requires host proteins also DNA replicated by a strand displacement mechanism No Okazaki fragments, continuous synthesis Protein TP acts as primer (attached to 5’ end) Example of class I: Adenovirus Example of class I: Herpesvirus

Enveloped icosahedral virus, ~ 200 nm in diameter Linear, double stranded DNA Example of class I: Herpesvirus

Utilizes host RNA polymerase and host RNA modiﬁcation enzymes A viral protein VP16 (tegument) enters the nucleus and binds to viral genome. This is part of the transcription factor complex recognized by host RNA polymerase. Example of class I: Herpesvirus

Early proteins encoded - DNA polymerase, DNA binding proteins, thymidine kinase, ribonucleotide reductase Comparatively more viral proteins utilized (drug targets) Precise mechanism of replication not known Newly synthesized DNA exists as concatamers, cleaved to appropriate size during packaging Example of class I: Poxvirus

Large enveloped virus, diameter ~ 200 nm Contains a double stranded DNA genome (192 kbp) Contains ~ 250 genes Example of class I: Poxvirus

Packages its own DNA dependent RNA polymerase Naked poxvirus DNA is not infectious at all Methylation, capping, polyadenylation of mRNA carried out by virally encoded and packaged enzymes One of the immediate early mRNAs codes for an “uncoating” protein Replication occurs in the cytoplasm, in “viral factories” Replication of DNA occurs by strand displacement and concatemer resolution Five major viral proteins are used, along with some cellular proteins Hepnadviruses – Class I/VII

Enveloped, icosahedral viruses containing a partially double stranded, circular, but not covalently closed DNA genome Examples - Hepatitis B virus Replicates through an RNA intermediate Packages its own DNA polymerase, which has reverse transcriptase activity Hepnadviruses – Class I/VII

cccDNA DNA repair RNA pol II transcription

cap pA Reverse pgRNA transcription

RC-DNA

Schematic of replication cycle Example of class IIa: Φx174

Parental genome

Parental RF

Rolling circle replication and formation of concatemers Example of class IIb: Parvoviruses

Small, non-enveloped, icosahedral, 18-25 nm in diameter Autonomous parvoviruses (MVM) use host cell enzymes for replication, package -ve stranded DNA Defective parvoviruses (AAV) need helper virus co-infection, Package -ve and +ve stranded DNA (in different virions) Contains palindromic terminal sequences which can serve as primers Replication proceeds by single strand displacement, no lagging strand synthesis. Replication strategy Replication scheme

Class IIa Class IIb +DNA ±DNA -DNA Class I

Class VI +RNA

Class IV mRNA -RNA Class V

±RNA

Baltimore classiﬁcation Class III Properties of RNA viruses

Genome usually smaller than that of DNA viruses Probably because of the higher rate of error accumulation in RNA genomes RNA dependent RNA polymerase necessary (no DNA stage) Relatively few proteins Properties of RNA viruses

Strategy to make multiple proteins - a) Producing multiple monocistronic RNAs b) Producing a primary transcript that is processed by host splicing machinery c) Producing a large polypeptide which is cleaved into individual proteins d) Allowing ribosomes to bind internally to viral RNA Example of Class IV – Poliovirus

Plus stranded RNA virus, functions as mRNA Infectious genome Replication proceeds in presence of DNA synthesis inhibitors, so no DNA intermediate Requires a RNA dependent RNA polymerase (translated initially) Example of Class IV – Poliovirus

Roy, J Gen Virol, 2008 Example of Class IV – Poliovirus

~ 10 times more +ve strand RNA produced +ve strand RNAs packaged into virions and removed as template quickly, -ve strand RNAs remain available as templates continuously IRES Genomic (+) RNA VPg AAAAA Polyprotein (includes polymerase) start codon stop codon for translation for translation

Genome corresponds to mRNA, polyadenylated, no methylated cap Contains internal ribosome binding site (IRES) Polyprotein produced, which is cleaved to generate RNA polymerase Example of Class IV – Poliovirus

VPg serves as primer for replication May be cleaved off and recycled by a host protein TBP2 “unlinkase” A lot of VPg and polymerase per cell! Needs to replicate in vesicles IRES Genomic (+) RNA VPg AAAAA Polyprotein (includes polymerase) start codon stop codon for translation for translation

structural proteins non-structural proteins 5’ VPg VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D VP4 3’

3C Replication scheme of poliovirus RNA SS RI SS RI + SS ’ ’ ’ ’ 5’ + 5 +5 3 3 +

+ +

polymerase

RF RF

Poliovirus RNA dependent RNA polymerase

Hobson et al, EMBO J, 2001 Poliovirus RNA dependent RNA polymerase

RNA polymerase activity is highly cooperative Interaction via interface I – necessary for binding RNA Interaction via interface II – necessary for catalysis

Hobson et al, EMBO J, 2001 Replication scheme of Qbeta RNA

~ 10 times more +ve strand RNA produced +ve strand RNAs packaged into virions and removed as template quickly, -ve strand RNAs remain available as templates continuously RNA dependent RNA polymerase consists of four host encoded and one virus encoded polypeptide Very speciﬁc for Qbeta RNA Complete Qbeta polymerase

Polypeptide Source MW (KD) function

Binding to + strand Ribosomal 70 Protein S1

Initiation Elongation factor 45 Tu

Initiation Elongation factor 35 Ts

Chain elongation Qbeta encoded 65

Minus strand Ribosome 72 synthesis associated (hexameric protein) Poliovirus replication occurs in large cytoplasmic vesicles

Uninfected cell Infected cell

Host factors are important

Doedens et al, J Virol, 2001 Class V RNA viruses

Minus stranded RNA virus Non-Infectious genome Requires a RNA dependent RNA polymerase, which is packaged in the virus Initial event after infection is synthesis of plus stranded RNA Examples – orthomyxoviruses (inﬂuenza), Paramyxoviruses (measles), Rhabdoviruses (rabies) Example of class V - Rhabdovirus + + + + + polymerase and + other proteins

- - + -

+ - + - + - Virion RNA + -

polymerase Example of class V - Rhabdovirus

Occurs in the cytosol Synthesis of mRNAs can occur from partially packaged nucleocapsids mRNA modiﬁcation etc carried out by packaged genome 5 proteins are synthesized - N, P, M, G, L New copies of -ve strand RNA are coated with N Example of class V – Inﬂuenza virus

Contains a fragmented genome - 8 -ve strand RNA wrapped with nucleocapsid mRNA synthesis and replication occurs in the nucleus with viral polymerase Unique mechanism for capping the mRNAs Nascent strands immediately wrapped with nucleocapsid Synthesis of inﬂuenza virus mRNAs

Cleavage site

Host mRNA A cap Viral exonuclease A

A Inﬂuenza virus nucleocapsid U

Viral polymerase

A Viral mRNA U Inﬂuenza virus RNA polymerase

Three subunits - PB1, PB2, PA PB1 recognizes cellular mRNA caps, has exonuclease activity PB2 elongates the cleaved primer PA involved in synthesis of -ve strand RNAs Replication scheme of reoviruses

Entire cycle occurs in the cytoplasm Reovirus partially uncoats into cores (infectious) Example of class III - Reovirus

Double stranded RNA virus, contains 10 segments of ds RNA Requires a RNA dependent RNA polymerase, which is packaged in the virus First step is to make mRNA - produced by viral polymerase and modiﬁed by virally encoded proteins in the “core” mRNA extruded through channels at the 5-fold axes of symmetry of the particles Old (input) mRNA is conserved inside cores, serve as templates New +ve strand RNA packaged into progeny virions, where the complementary strand is synthesized Replication scheme of reoviruses + + -

assortment mRNA Core of +ve RNA

+ - proteins

new virion Schematic of bluetongue virus (orbivirus) core

VP3 and VP7 form the core surface

Roy, J Gen Virol, 2008 BTV core VP7

VP3 VP1+VP4

Roy, J Gen Virol, 2008 Example of class VI - HIV

Plus stranded RNA virus, contains 2 RNA molecules with 5’cap and 3’ polyA tail Physically linked through hydrogen bonds Not released into the cytosol, cannot function as mRNA Non-infectious genome Replication cannot proceed in presence of DNA synthesis inhibitors (actinomycin D) Example of class VI - HIV

An RNA dependent DNA polymerase (reverse transcriptase) is used to convert the ssRNA genome into dsDNA The existence of the enzyme ﬁrst proposed by Howard Temin Enzyme isolated by Temin and Baltimore Mistake prone enzyme - 1 mistake/20,000 bases, contains Rnase H, Integrase and protease activities, utilizes tRNA as primer Enzyme and tRNA packaged into the particles along with genome Example of class VI - HIV

Example of class VI - HIV

R U5 Leader Coding area U3 R Cap A(n) PBS PPT R - terminally redundant region U3, U5 - unique, non-coding region, form 5’ and 3’ end of provirus genome, contains promoter elements for provirus transcription PBS - primer binding site Leader region - non-coding, present at 5’ of viral mRNAs PPT - polypurine tract, initiates +ve strand synthesis during reverse transcription Example of class VI - HIV

R U5 Leader Coding area U3 R Cap A(n) PBS PPT

LTR LTR U3 R U5 U3 R U5

Viral dsDNA

Integrated into host genome by Integrase Expression of HIV mRNA

Transcription by host RNA polymerase II Utilize splicing and ribosomal frameshifting to make proteins Tat - transcription activator protein Rev - Helps in the export of mRNA and genomic RNA to the cytosol